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PubMed2001

Phosphorylation of the myosin-binding subunit of myosin phosphatase by Raf-1 and inhibition of phosphatase activity.

Raf-1 serine/threonine protein kinase plays an important role in cell survival, proliferation, and migration; however, the specific targets of Raf-1 in diverse cellular processes are not clearly defined. Myosin phosphatase activity is critical to the regulation of cytoskeletal reorganization, cytokinesis, and cell motility. Here, we describe the association of Raf-1 with myosin phosphatase and phosphorylation of the regulatory myosin-binding subunit (MBS) of myosin phosphatase by Raf-1. Treatment of cells with phorbol 12-myristate 13-acetate has been shown to stimulate Raf-1 protein kinase. To determine the effect of enzymatic activation of Raf-1 on MBS phosphorylation, COS-1 cells were transiently transfected with FLAG-tagged full-length Raf-1. A significantly higher phosphorylation of purified glutathione S-transferase-tagged truncated MBS protein (amino acids 654-880) occurred in the presence of FLAG-Raf-1 immunoprecipitated from phorbol 12-myristate 13-acetate-treated cells compared with untreated cells ( approximately 3.0-fold). Using a sequential kinase-phosphatase assay and phosphorylated myosin light chain as substrate in the phosphatase reaction, we showed that Raf-1-associated protein phosphatase-specific activity was inhibited (relative phosphatase activity without and with adenosine 5'-O-(3-thiotriphosphate): 100 and approximately 30%, respectively). Previously, ionizing radiation has been shown to activate Raf-1 (Kasid, U., Suy, S., Dent, P., Ray, S., Whiteside, T. L., and Sturgill, T. W. (1996) Nature 382, 813-816). Exposure of cells to ionizing radiation resulted in the increased association of Raf-1 with MBS (3-6-fold versus unirradiated control) and inhibition of Raf-1-associated protein phosphatase-specific activity (relative phosphatase activity without and with ionizing radiation: 100 and approximately 54%, respectively). Our studies identify MBS as a new substrate of Raf-1 and implicate a role for Raf-1 in the regulation of pathways involving myosin phosphatase activity.

Animals↗

PubMed1984

The protein phosphatases involved in cellular regulation. Identification of the inhibitor-2 phosphatases in rabbit skeletal muscle.

Inhibitor-2, purified by an improved procedure, was used to identify protein phosphatases capable of catalysing its dephosphorylation. The results showed that, under our experimental conditions, protein phosphatases-1, 2A and 2B were the only significant protein phosphatases in rabbit skeletal muscle extracts acting on this substrate. Protein phosphatases-1 and 2A accounted for all the inhibitor-2 phosphatase activity in the absence of Ca2+ (resting muscle), and the potential importance of these enzymes in vivo is discussed. Protein phosphatase-2B, a Ca2+-calmodulin-dependent enzyme, could account for up to 30% of the inhibitor-2 phosphatase activity in contracting muscle. The Km of protein phosphatase-1 for inhibitor-2 (40 nM) was 100-fold lower than the Km for phosphorylase a (4.8 microM). This finding, coupled with the failure of inhibitor-2 to inhibit its own dephosphorylation, suggests that inhibitor-2 is dephosphorylated at one of the two sites on protein phosphatase-1 involved in preventing the dephosphorylation of other substrates. The dephosphorylation of inhibitor-2 by protein phosphatase-1 was also unaffected by inhibitor-1, suggesting that the phosphorylation state of inhibitor-2 is unlikely to be controlled by cyclic AMP in vivo.

Animals↗

PubMed1985

The protein phosphatases involved in cellular regulation. Purification and characterisation of the glycogen-bound form of protein phosphatase-1 from rabbit skeletal muscle.

A type-1 protein phosphatase (protein phosphatase-1G) was purified to homogeneity from the glycogen-protein particle of rabbit skeletal muscle. Approximately 3 mg of enzyme were isolated within 4 days from 5000 g of muscle. Protein phosphatase-1G had a molecular mass of 137 kDa and was composed of two subunits G (103 kDa) and C (37 kDa) in a 1:1 molar ratio. The subunits could be dissociated by incubation in the presence of 2 M NaCl, separated by gel-filtration on Sephadex G-100, and recombined at low ionic strength. The C component was the catalytic subunit, and was identical to the 37-kDa type-1 protein phosphatase catalytic subunit (protein phosphatase-1C) isolated from ethanol-treated muscle extracts, as judged by peptide mapping. The G component was the glycogen-binding subunit. It was very asymmetric, extremely sensitive to proteolytic degradation, and failed to silver stain on SDS/polyacrylamide gels. Protein phosphatase-1G was inhibited by inhibitor-1 and inhibitor-2, but unlike protein phosphatase-1C, the rate of inactivation was critically dependent on the ionic strength, temperature and time of preincubation with the inhibitor protein. At near physiological temperature and ionic strength, protein phosphatase-1G was inactivated very rapidly by inhibitor-1. Protein phosphatase-1G interacted with inhibitor-2 (I-2) to form an inactive species, with the structure GCI-2. This form could be activated by preincubation with Mg-ATP and glycogen synthase kinase-3. The G subunit could be phosphorylated on a serine residue(s) by cyclic-AMP-dependent protein kinase, but not by phosphorylase kinase or glycogen synthase kinase-3. Phosphorylation was rapid and stoichiometric, and increased the rate of inactivation of protein phosphatase-1G by inhibitor-1. The relationship of the G subunit to the 'deinhibitor protein' is discussed.

Animals↗

PubMed1990

Regulation of norepinephrine secretion in permeabilized PC12 cells by Ca2(+)-stimulated phosphorylation. Effects of protein phosphatases and phosphatase inhibitors.

Protein phosphatases and phosphatase inhibitors were used to examine the role of protein phosphorylation in the regulation of norepinephrine secretion in digitonin-permeabilized PC12 cells. The addition of an exogenous type 2A protein phosphatase caused as much as a 70% decrease in Ca2(+)-dependent norepinephrine secretion. In the presence of okadaic acid, a potent inhibitor of type 2A protein phosphatases, phosphatase 2A had no effect on secretion. The addition of exogenous calcineurin, a Ca2(+)-calmodulin-stimulated phosphatase, also caused decrease in Ca2(+)-dependent secretion, but on a molar basis it was less effective than phosphatase 2A. Two phosphatase inhibitors, 1-naphthylphosphate and sodium pyrophosphate, caused 75-100% increases in the amount of norepinephrine secreted in the absence of Ca2+ without affecting the amount of norepinephrine secreted in the presence of Ca2+. This stimulation of Ca2(+)-independent secretion by 1-naphthylphosphate and pyrophosphate suggests that there is a slow rate of Ca2(+)-independent phosphorylation and that phosphorylation triggers secretion. Unlike the results obtained in the presence of ATP, secretion in the presence of adenosine-5'-O-(3-thiotriphosphate), ATP gamma S, was not affected by the addition of type 2A protein phosphatase or by the addition of phosphatase inhibitors. These results are consistent with secretion in these permeabilized cells being regulated by a Ca2(+)-stimulated phosphorylation.

Adenosine Triphosphate↗

PubMed1981

Subcellular localisation and properties of histone phosphate phosphatase in human polymorphonuclear leukocytes: alterations in pregnancy and chronic granulocytic leukaemia and relationship to alkaline phosphatase.

Using [32P]histone as substrate, an assay for histone phosphate phosphatase was optimised for human polymorphonuclear leukocytes. Kinetic studies showed that the activity was optimal at pH 6.8, was stimulated by Mn2+ and Co2+, and inhibited by sodium sulphite and zinc chloride. The apparent Km of the enzyme for histone phosphate was 0.89 mumol/l. Neutrophils were homogenized in isotonic sucrose and, after low speed centrifugation, the supernatant was subjected to analytical subcellular fractionation. Gradient fractions were assayed for principal marker enzymes and for histone phosphate phosphatase. Histone phosphate phosphatase activity was shown to be solely located to the cytosol. No activity was detected in the alkaline phosphatase-containing granules. Neutrophils were isolated from the blood of control subjects, patients with chronic granulocytic leukaemia and women in the third trimester of pregnancy. The specific activity (milliunits/mg protein) of histone phosphate phosphatase was significantly reduced in patients with chronic granulocytic leukaemia compared to control values but this decrease was considerably less than that found for alkaline phosphatase. The possible implication of the reduced histone phosphatase activity in leukaemia neutrophils is discussed. There was no significant change in histone phosphate phosphatase in leucocytes from pregnant women. These results, together with the subcellular fractionation experiments and inhibitor studies, strongly indicate that histone phosphate phosphatase is not attributable to neutrophil alkaline phosphatase.

Alkaline Phosphatase↗

PubMed1984

Studies of the biochemical and immunological properties of human neutrophil alkaline phosphatase with comparison to the established alkaline phosphatase isoenzymes.

A range of affinity column chromatographic procedures and various inhibitors have been used to compare human neutrophil alkaline phosphatase with the three established isoenzymes. The column chromatography studies have clearly distinguished neutrophil alkaline phosphatase from the intestinal isoenzyme. Inhibition studies with L-phenylalanine, L-homoarginine and levamisole have revealed a distinct pattern of inhibition for liver, kidney and neutrophil alkaline phosphatase which is quite different from the pattern shown by placental and intestinal alkaline phosphatase. Immunospecificity experiments with a monoclonal antibody raised to human liver alkaline phosphatase have shown that it cross reacts with alkaline phosphatase from kidney, bone and neutrophil. In all studies, neutrophil alkaline phosphatase has virtually identical properties to that of liver, kidney and bone alkaline phosphatase. This is strong evidence that neutrophil alkaline phosphatase is a product of the same structural gene which codes for the liver/bone/kidney group of human alkaline phosphatases.

Alkaline Phosphatase↗

PubMed1997

Phosphatidate phosphatases and diacylglycerol pyrophosphate phosphatases in Saccharomyces cerevisiae and Escherichia coli.

Phosphatidate phosphatase plays a major role in the synthesis of phospholipids and triacylglycerols in the yeast Saccharomyces cerevisiae. Membrane- and cytosolic-associated forms of the enzyme have been isolated and characterized. These enzymes are Mg2+-dependent and N-ethylmaleimide-sensitive. The expression of a membrane-associated form of phosphatidate phosphatase is regulated by growth phase and inositol supplementation, whereas enzyme activity is regulated by lipids, nucleotides, and by phosphorylation. Phosphatidate phosphatase is coordinately regulated with other phospholipid biosynthetic enzymes including phosphatidylserine synthase. Diacylglycerol pyrophosphate phosphatase is a novel enzyme of phospholipid metabolism which is present in S. cerevisiae, Escherichia coli, and mammalian cells. This enzyme possesses a phosphatidate phosphatase activity which is Mg2+-independent and N-ethylmaleimide-insensitive and is distinct from the Mg2+-dependent and N-ethylmaleimide-sensitive form of phosphatidate phosphatase. Genes encoding for diacylglycerol pyrophosphate phosphatase have been isolated from S. cerevisiae and E. coli. The deduced protein sequences of these genes show homology to the sequence of the mouse PAP2 (Mg2+-independent and N-ethylmaleimide-insensitive phosphatidate phosphatase) protein, especially in a novel phosphatase sequence motif. Rat liver PAP2 displays diacylglycerol pyrophosphate phosphatase activity.

Amino Acid Sequence↗

PubMed2001

Alkaline fixation-resistant acid phosphatases in human tissues: histochemical evidence for a new type of acid phosphatase in endothelial, endometrial and neuronal sites.

The effect of pH during formalin fixation on acid phosphatases in human tissues was studied. Lysosomal-type acid phosphatase was sensitive to alkaline fixation, being completely inactive after fixation at pH 9.0. Prostatic and tartrate-resistant osteoclastic/macrophagic types were alkaline fixation-resistant, as was an acid phosphatase localized in endothelium, endometrial stromal cells and intestinal nerves. The latter activity was further separable into fluoride- and tartrate-sensitive beta-glycerophosphatase and fluoride-sensitive, tartrate-resistant alpha-naphthyl phosphatase. The activities appeared to represent either different, tightly associated enzymes or separate activity centres of a single enzyme. Alkaline fixation-resistant alpha-naphthyl phosphatase at endothelial, endometrial and neuronal sites was also well demonstrated in unfixed or neutral formalin-fixed sections as tartrate-resistant activity similar to classical tartrate-resistant acid phosphatase, but these phosphatases appear to be antigenically different. Alkaline fixation-resistant acid phosphatase showed a restricted tissue distribution both in endothelium (mainly in vessels of abdominal organs) and at neuronal sites (only in intestinal nerves). Alkaline fixation-resistant acid phosphatase appears to represent a previously unknown or uncharacterized enzyme activity whose chemical properties could not be classified as any previously known type of acid or other phosphatases.

Acid Phosphatase↗

PubMed1997

Mammalian Mg2+-independent phosphatidate phosphatase (PAP2) displays diacylglycerol pyrophosphate phosphatase activity.

Recent studies indicate that the metabolism of diacylglycerol pyrophosphate (DGPP) is involved in a novel lipid signaling pathway. DGPP phosphatases (DGPP phosphohydrolase) from Saccharomyces cerevisiae and Escherichia coli catalyze the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyze the dephosphorylation of PA to yield diacylglycerol. We demonstrated that the Mg2+-independent form of PA phosphatase (PA phosphohydrolase, PAP2) purified from rat liver catalyzed the dephosphorylation of DGPP. This reaction was Mg2+-independent, insensitive to inhibition by N-ethylmaleimide and bromoenol lactone, and inhibited by Mn2+ ions. PAP2 exhibited a high affinity for DGPP (Km = 0.04 mol %). The specificity constant (Vmax/Km) for DGPP was 1. 3-fold higher than that of PA. DGPP inhibited the ability of PAP2 to dephosphorylate PA, and PA inhibited the dephosphorylation of DGPP. Like rat liver PAP2, the Mg2+-independent PA phosphatase activity of DGPP phosphatase purified from S. cerevisiae was inhibited by lyso-PA, sphingosine 1-phosphate, and ceramide 1-phosphate. Mouse PAP2 showed homology to DGPP phosphatases from S. cerevisiae and E. coli, especially in localized regions that constitute a novel phosphatase sequence motif. Collectively, our work indicated that rat liver PAP2 is a member of a phosphatase family that includes DGPP phosphatases from S. cerevisiae and E. coli. We propose a model in which the phosphatase activities of rat liver PAP2 and the DGPP phosphatase of S. cerevisiae regulate the cellular levels of DGPP, PA, and diacylglycerol.

Amino Acid Sequence↗

PubMed1999

Evidence for a calpeptin-sensitive protein-tyrosine phosphatase upstream of the small GTPase Rho. A novel role for the calpain inhibitor calpeptin in the inhibition of protein-tyrosine phosphatases.

Activation of the thiol protease calpain results in proteolysis of focal adhesion-associated proteins and severing of cytoskeletal-integrin links. We employed a commonly used inhibitor of calpain, calpeptin, to examine a role for this protease in the reorganization of the cytoskeleton under a variety of conditions. Calpeptin induced stress fiber formation in both forskolin-treated REF-52 fibroblasts and serum-starved Swiss 3T3 fibroblasts. Surprisingly, calpeptin was the only calpain inhibitor of several tested with the ability to induce these effects, suggesting that calpeptin may act on targets besides calpain. Here we show that calpeptin inhibits tyrosine phosphatases, enhancing tyrosine phosphorylation particularly of paxillin. Calpeptin preferentially inhibits membrane-associated phosphatase activity. Consistent with this observation, in vitro phosphatase assays using purified glutathione S-transferase fusion proteins demonstrated a preference for the transmembrane protein-tyrosine phosphatase-alpha over the cytosolic protein-tyrosine phosphatase-1B. Furthermore, unlike wide spectrum inhibitors of tyrosine phosphatases such as pervanadate, calpeptin appeared to inhibit a subset of phosphatases. Calpeptin-induced assembly of stress fibers was inhibited by botulinum toxin C3, indicating that calpeptin is acting on a phosphatase upstream of the small GTPase Rho, a protein that controls stress fiber and focal adhesion assembly. Not only does this work reveal that calpeptin is an inhibitor of protein-tyrosine phosphatases, but it suggests that calpeptin will be a valuable tool to identify the phosphatase activity upstream of Rho.

3T3 Cells↗

PubMed2004

Phosphate binding in the active site of alkaline phosphatase and the interactions of 2-nitrosoacetophenone with alkaline phosphatase-induced small structural changes.

To monitor structural changes during the binding of Pi to the active site of mammalian alkaline phosphatase in water medium, reaction-induced infrared spectroscopy was used. The interaction of Pi with alkaline phosphatase was triggered by a photorelease of ATP from the inactive P(3)-[1-(2-nitrophenyl)]ethyl ester of ATP. After photorelease, ATP was sequentially hydrolyzed by alkaline phosphatase giving rise to adenosine and three Pi. Although a phosphodiesterase activity was detected prior the photorelease of ATP, it was possible to monitor the structural effects induced by Pi binding to alkaline phosphatase. Interactions of Pi with alkaline phosphatase were evidenced by weak infrared changes around 1631 and at 1639 cm(-1), suggesting a small distortion of peptide carbonyl backbone. This result indicates that the motion required for the formation of the enzyme-phosphate complex is minimal on the part of alkaline phosphatase, consistent with alkaline phosphatase being an almost perfect enzyme. Photoproduct 2-nitrosoacetophenone may bind to alkaline phosphatase in a site other than the active site of bovine intestinal alkaline phosphatase and than the uncompetitive binding site of L-Phe in bovine intestinal alkaline phosphatase, affecting one-two amino acid residues.

Adenosine Triphosphate↗

PubMed1983

Characterization of a phosphotyrosyl protein phosphatase activity associated with a phosphoseryl protein phosphatase of Mr = 95,000 from bovine heart.

A cytosolic phosphoprotein phosphatase of Mr = 95,000 purified from bovine cardiac muscle, which contains a catalytic subunit of Mr = 35,000, is known to be associated with a Mg2+-activated p-nitrophenyl phosphatase activity. We have found that the enzyme preparation is also active toward phosphotyrosyl-IgG and -casein phosphorylated by pp60v-src, the transforming gene product of Rous sarcoma virus. The properties of this phosphotyrosyl protein phosphatase activity closely resemble those of the p-nitrophenyl phosphatase activity but sharply differ from those of the phosphorylase phosphatase activity. Comparative studies of the activities of the Mr = 95,000 phosphatase, bovine kidney alkaline phosphatase, and ATP X Mg-dependent phosphatase toward phosphoseryl, phosphothreonyl, and phosphotyrosyl proteins and p-nitrophenyl phosphate under various conditions have been carried out. The results indicate that the Mr = 95,000 enzyme exhibits higher activity toward phosphoseryl and phosphothreonyl proteins than toward phosphotyrosyl proteins, while the kidney alkaline phosphatase preferentially dephosphorylates phosphotyrosyl proteins. ATP X Mg-dependent phosphatase is inactive toward phosphotyrosyl proteins.

Animals↗

PubMed1983

Macromolecular alkaline phosphatase and an immunoglobulin G that inhibited alkaline phosphatase in a patient's serum.

Macromolecular alkaline phosphatase (EC 3.1.3.1) was found in the serum of a patient suffering from myasthenia gravis (adult type II) complicated with thymoma, and was shown by immunoelectrophoresis to be bound to immunoglobulins A and G (IgG). Placental alkaline phosphatase, complexed with either the patient's serum or IgG purified from the patient's serum, remained at the origin on electrophoresis, with significant loss of activity. Intestinal alkaline phosphatase, complexed with either the patient's serum or the patient's IgG, migrated to a position similar to that of the macromolecular alkaline phosphatase in the patient's serum on electrophoresis. About 50% of the placental alkaline phosphatase activity was inhibited with 0.1-0.2 g of the patient's IgG per liter, but 6.93 g of the IgG per liter was required for about 20% inhibition of the intestinal alkaline phosphatase activity. The complex of intestinal alkaline phosphatase with the patient's IgG was fairly heat stable. From these results, we concluded that the macromolecular alkaline phosphatase in the patient's serum consisted of intestinal alkaline phosphatase and IgG that was specific for placental alkaline phosphatase.

Adult↗

PubMed1985

Acid phosphatase and phosphoamino acid phosphatases in murine erythroleukaemic cells.

The activities of acid and alkaline phosphatases and phosphotyrosine, phosphoserine and phosphothreonine phosphatases were measured in Friend murine erythroleukaemic (MEL) cells. The effects of treating the cells with dimethyl sulphoxide (DMSO), an inducer of differentiation, were examined. In untreated cells alkaline phosphatase activity was undetectable, though there were significant amounts of acid phosphatase (76 +/- 15 mU/mg protein) and phosphotyrosine phosphatase (16 +/- 0.9mU/mg protein); phosphoserine and phosphothreonine phosphatase activities (9 +/- 0.4 and 7 +/- 0.6mU/mg protein, respectively) were lower than for phosphotyrosine phosphatase. Addition of 1 or 2% DMSO to the culture medium resulted in the expected cell death within 2 weeks. With 0.5% DMSO, cells remained viable for at least 8 weeks, but while some appeared to have smaller nuclei and retained their rounded appearance, others became fibroblastic within several days and adhered to the culture vessel. The treated cells which had kept their morphology showed no difference in acid phosphatase activities as compared with untreated controls; phosphotyrosine phosphatase was lower (9 +/- 0.8mU/mg protein) and phosphoserine and phosphothreonine phophatases higher (11 +/- 0.5 and 10 +/- 0.4mU/mg protein, respectively) than in the controls. The Km values for p-nitrophenyl phosphate were similar in untreated and treated cells (0.069 and 0.068mM, respectively); for phosphotyrosine the Km value was lower in the treated cells (0.97mM) than in the controls (1.9mM).

Acid Phosphatase↗

PubMed1988

Histone H1 phosphorylated by protein kinase C is a selective substrate for the assay of protein phosphatase 2A in the presence of phosphatase 1.

A protein phosphatase assay, selective for protein phosphatase 2A, has been developed. Bovine histone H1 phosphorylated by protein kinase C and [gamma-32P]ATP, designated H1(C), was tested as the substrate for various preparations of protein phosphatases 1 and 2A. The phosphatase 2A preparations were 10-60-times more active with H1(C) as the substrate when compared to phosphorylase a. The phosphatase 1 enzymes showed very little dephosphorylation of the H1(C) substrate, the activity being less than 5% of the phosphorylase phosphatase activity. This preference and selectivity was demonstrated for purified phosphatase preparations in addition to fresh tissue extracts. The assay provides a rapid, simple assay for the routine analysis of phosphatase 2A in the presence of phosphatase 1, without the use of heat-stable inhibitor proteins.

Animals↗

PubMed2003

Elevated levels of protein phosphatase 1 and phosphatase 2A may contribute to cardiac dysfunction in diabetes.

Although protein phosphorylation and dephosphorylation are known to regulate the activities of different enzymes, sufficient information on the role of dephosphorylation in cardiac function is not available. Since protein phosphatases mediate dephosphorylation, it is possible that cardiac dysfunction induced by diabetes may be due to alterations in the activities of these enzymes. We therefore determined cardiac protein phosphatase activity as well as protein contents of phosphatase 1 and phosphatase 2A in diabetic animals. For this purpose, rats were made diabetic by administering a single intravenous injection of streptozotocin (65 mg/kg body weight) and hearts were examined after 1, 2, 3, 4 and 8 weeks. Some of the 4-week diabetic animals received subcutaneous injections of insulin (3 U/day) for a further period of 4 weeks. Cardiac dysfunction was evident after 2 weeks of inducing diabetes and deteriorated further with time. A significant increase in protein phosphatase activity appeared after 1 week and persisted until 8 weeks. Increased protein phosphatase activity in the diabetic heart was associated with a corresponding increase in the protein contents of both phosphatase 1 and phosphatase 2A. Insulin treatment partly prevented the changes observed in diabetic animals. The results suggest that increased protein phosphatase activities and subsequent enhanced protein dephosphorylation may play a role in diabetes-induced cardiac dysfunction.

Animals↗

PubMed1992

Protein phosphatase 2A is a specific protamine-kinase-inactivating phosphatase.

Purified preparations of a protamine protein kinase from bovine kidney cytosol [Damuni, Amick & Sneed (1989) J. Biol. Chem. 264, 6412-6416] were inactivated after incubation with near-homogeneous preparations of protein phosphatase 2A1 and protein phosphatase 2A2. These protein phosphatase 2A-mediated inactivations of the protamine kinase were unaffected by highly purified preparations of inhibitor 2, but were prevented when the incubations were performed in the presence of 100 nM microcystin-LR, 100 nM okadaic acid or 0.2 mM-ATP. By contrast, highly purified preparations of protein phosphatase 2B, protein phosphatase 2C, the catalytic subunit of protein phosphatase 1, and two forms of a protein tyrosine phosphatase, designated PTPase 1B and T-cell PTPase, had little effect, if any, on protamine kinase activity. Purified preparations of the protamine kinase did not react with anti-phosphotyrosine antibodies, as determined by Western blotting and immunoprecipitation analysis. The results indicate that protein phosphatase 2A is a specific protamine-kinase-inactivating phosphatase.

Animals↗

PubMed1982

Dolichyl-phosphate phosphatase and dolichyl-diphosphate phosphatase in rat-liver microsomes.

Dolichyl-phosphate phosphatase and dolichyl-diphosphate phosphatase activities of a liver-cell microsomal preparation were solubilized by treatment with Triton X-100. The 100,000 X g supernatant was then passed through a column of Sepharose-4B--concanavalin A. Both enzyme activities were found in the percolate. This treatment eliminated inhibition by ATP and glucose 6-phosphate in both phosphatase activities. In each case the activities were inhibited by higher concentrations of enzyme preparation due to the presence of phospholipids. The inhibitory effects of either phosphatidylcholine or phosphatidylethanolamine were due to competition for detergent. On the other hand, the effect produced by phosphatidic acid appeared to be different, since it did not change the optimal concentration of Triton X-100 for the two enzymes. Dolichyl-phosphate phosphatase was strongly inhibited by both Pi and PPi, whereas dolichyl-diphosphate phosphatase was only slightly inhibited by Pi and not at all by PPi. Dolichyl-diphosphate phosphatase was more inhibited by divalent cations than dolichyl-phosphate phosphatase. The apparent Km of dolichyl-phosphate phosphatase for dolichyl phosphate was 0.15 mM. Dolichol also inhibited dolichyl-phosphate phosphatase, but it produced a stronger inhibition on dolichyl-diphosphate phosphatase. The inhibitory effect of dolichol was not entirely due to detergent competition.

Animals↗